The field of this invention is novel peptides useful for modulating the activity of immune system cells and for the inhibition of inflammation.
The immune system is an extraordinarily complex combination of cells and compositions that protects a mammalian host against a wide variety of pathogens, while surveiling the body against deleterious aberrations, such as neoplasia. One branch of the immune system involves the cells that carry out immune system functions, including both (a) lymphocytes, such as the bone marrow-derived B-lymphocytes, the thymus-derived T lymphocytes and natural-killer (NK) cells, and (b) the mononuclear phagocytes, including both monocytes and macrophages. While lymphocytes are primarily associated with specific immune responses, due to their ability to specifically recognize and distinguish antigenic determinants, the mononuclear phagocytes are most often involved in the general removal of foreign microbes through phagocytosis as well as the production and secretion of cytokines as induced either directly by a microbe itself or in response to antigen-stimulated T lymphocytes. The functions of lymphocytic cells and the mononuclear phagocytes are highly interconnected and essential for proper immune system function.
One important subset of lymphocytic cells are T lymphocytes, which derive their designation from the fact that they are processed by the thymus. T lymphocytes are a complex group of cells which may be cytotoxic, having numerous mechanisms for inducing cell death, or activating, by secreting varous cytokines that function to activate other cells. Cytotoxic T lymphocytes (xe2x80x9cCTLsxe2x80x9d) act by being restricted to a particular major histocompatibility complex (MHC) antigen and express a cell surface T cell receptor which comprises both an xcex1 and xcex2 chain and which has specific affinity for a particular MHC complex associated with a peptide in the groove of the MHC. CTLs have been screened so that they do not normally act against cells where the peptide in the groove is endogenous to the host. However, where the MHC is foreign or the peptide in the groove is foreign to the host, the CTLs will attack such cell and kill it. Other lymphocytic cells which play important roles in the immune response include B-lymphocytes and natural killer (NK) cells, both of whose activity may be influenced by other cells of the immune system and various cytokine polypeptides.
The mononuclear phagocytes constitute a second major cell population of the immune system and consist of cells having a common lineage whose primary function is phagocytosis. The mononuclear phagocytes derive from progenitor bone marrow stem cells and, after maturation and subsequent activation, can achieve various morphological forms, including incompletely differentiated monocyte cells and macrophages. Proper function of the mononuclear phagocytes is dependent on the ability to both produce and respond to various cytokine proteins.
Cytokines, such as the various interferons, interleukins, tumor necrosis factors, chemokines, hematopoietic growth factors and migration inhibition factors are a diverse group of proteins that are produced by a wide variety of different cells types of the immune system. Most importantly, cytokines are produced and/or responded to by various lymphocytes and mononuclear phagocytes in response to various stimuli. For the most part, cytokines are produced during the effector phases of both natural and specific immunity and serve to mediate and regulate both immune and inflammatory responses. Cytokines, like other polypeptide hormones, initiate their action by binding to specific receptors on the surface of target cells, their activation often resulting in an inflammatory response.
While activation of the immune response and cytokine-induced inflammatory responses are extremely important to a host""s health and proper functioning of the immune system, there are a number of situations where such activation is undesired. One particular area is associated with transplantation, where one rarely has an identical match between the donor and recipient of the MHC antigens. Another incidence is where there is a failure on the part of CTLs in that they attack cells where the MHC and associated peptide are both endogenous, as occurs in autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). An additional incidence is where a cytokine-mediated inflammatory response functions to adversely affect the health of the host, such as inflammatory responses associated with such maladies as septic shock, rheumatoid arthritis, Crohn""s disease, colitis, and the like.
Immunosuppression has become a general approach in situations where activation of CTLs is undesired. However, immunosuppressants such as cyclosporin A, FK506, and the like, have numerous undesirable side effects. Additionally, various approaches have been employed for controlling or inhibiting inflammatory responses, however, many of these approaches also have one or more undesirable effects. There is, therefore, substantial interest in identifying new agents which can act to inhibit the activation of lymphocytic cells, particularly CTLs, while having less of a universal immunosuppressive effect on the immune system and fewer side effects, so as to leave the host with a substantial proportion of the immune system for protection against adventitious infection. There is also a substantial interest in identifying new agents that function to control or inhibit adverse inflammatory reactions.
In the last few years, oligopeptides have been reported as being effective in modulating immune system activity and extending the lifetime of allogeneic transplants. These oligopeptides are based on the human leukocyte antigen-B (HLA-B) xcex11-domain and have a conserved amino acid sequence Arg-X-X-X-Arg-X-X-X-X-Tyr, (SEQ ID NO: 1) with the various amino acids designated as X varying within a relatively few amino acids to retain activity (e.g., see WO 95/13288). The mechanism by which these oligopeptides effectuate their activity is not understood, particularly as to how they cooperate with subtherapeutic doses of cyclosporin to extend the lifetime of allogeneic transplants.
Also reported (Manolios, NOVEL PEPTIDE, PCT application, filed based on Australian application Nos. PN 0589 and PN 0590, Jan. 16, 1995) as having an effect on T cell mediated inflammation are oligopeptides of the formula:
A-B-C-D-E
wherein: A is absent or is 1 or 2 hydrophobic residues; B is a positively charged amino acid; C is a peptide consisting of from 3 to 5 hydrophobic amino acids; D is a positively charged amino acid; and E is absent or is up to 8 hydrophobic amino acids. The peptides that were synthesized are: Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 2); Met-Gly-Leu-Arg-Ile-Leu-Leu-Leu (SEQ ID NO: 3); Leu-Gly-Ile-Leu-Leu-Leu-Gly-Val (SEQ ID NO4); Leu-Asp-Ile-Leu-Leu-Leu-Gly-Val (SEQ ID NO: 5); Leu-Arg-Ile-Leu-Leu-Leu-Ile-Leu-Val (SEQ ID NO: 6); and Leu-Arg-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 7). The sequences are predicated on the sequence of a transmembrane sequence of TCR-xcex1. There is no support in this application that the peptides have a beneficial effect on extending transplantation lifetimes.
Buelow et al., Transplantation 59:649-654 (1995) and references cited therein. Manolios et al., Nature Medicine 3:84-88 (1997) describes oligopeptides derived by rational design which modulate T cell activity. WO 95/13288 by Clayberger et al. which describes peptides capable of modulating T cell activity. References describing methods for designing compounds by computer using structure activity relationships include Grassy et al., J. of Molecular Graphics 13:356-367 (1995); Haiech et al., J. of Molecular Graphics 13:46-48 (1995); Yasri et al., Protein Engineering 11: 959-976 (1996) and Ashton et al., Drug Discovery Today 1:71-78 (1996).
Cytomodulating peptides are provided which are capable of (1) modulating the activity of various immune system cells, particularly lymphocytic cells, more particularly CTLs, (2) inhibiting the production of inflammatory cytokines by cells capable of producing such cytokines, thereby being effective in the treatment of conditions associated with adverse inflammatory reactions, (3) modulating the activity of heme-containing enzymes and/or (4) delaying the onset of insulin-dependent diabetes mellitus (IDDM) in a host susceptible of having IDDM, where the peptides are based upon a design in accordance with a computer program. Exemplary of the compounds are oligopeptides comprising the sequence B-X-X-X-B-X-X-X-J-Tyr (SEQ ID NO: 8), where B is a basic amino acid, J is Gly, B, or an aliphatic hydrophobic amino acid of from 5-6 carbon atoms and X is any amino acid other than an aliphatic polar amino acid, where at least three Xs are the same aliphatic non-polar amino acid, dimers thereof and D-stereoisomers thereof, and wherein the amino acid sequence may be part of a ring. The peptides find use for inhibiting the activation of immune system lymphocytes, particularly cytotoxic lymphocytes, either by themselves or in conjunction with other immunosuppressant agents, particularly in extending the lifetime of transplants. The peptides described herein also find use for inhibiting the production of inflammatory cytokines (e.g., interferon-xcex3, IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, MIP1xcex1, etc.), thereby being useful for inhibiting inflammatory responses associated with various disorders such as rheumatoid arthritis, septic shock, Crohn""s disease, colitis, allergic reactions, autoimmune diseases, and the like, for inhibiting the activity of heme-based enzymes such as heme oxygenase, nitric oxide synthase, etc., and delaying the onset of IDDM in a patient at risk for developing IDDM, both in vitro and in vivo. Administration of the peptides may be ex vivo of an organ to be transplanted or in vivo by any convenient means, including by direct application or administration of the peptide or nucleic acid encoding the desired peptide, in sufficient amount to substantially inhibit lymphocytic activation, inhibit the production of inflammatory cytokines and the associated inflammatory process, inhibit heme-based enzyme activity, an activity that has been previously associated with inflammatory responses and/or delaying the onset of IDDM.